Eastern equine encephalitis virus (EEEV), an Alphavirus in the family Togaviridae, is classified in Category B of the NIH Priority Pathogens List, and as a high consequence livestock pathogen by the USDA because it is highly lethal for humans and equines, and because effective vaccines and therapies are lacking, EEEV. The formalin-inactivated vaccine strain of EEEV is not suitable for wide-scale human use due to poor immunogenicity and possible residual virulence. Clearly alternative strategies for vaccine production are required. Our long-term goal is to develop a live-attenuated virus vaccine with sufficient degree of attenuation to be safe for human populations. The objective of the proposed research is the rational design of attenuated strains via the selective deletion (or disabling) of innate immune evasion properties. This will be based on the hypothesis that EEEV possesses mechanism(s) to antagonize the interferon alpha/beta (IFN-a/b) response elicited by infected dendritic cells (DCs) which can be disabled to attenuate the virus and enhance the immune response. Several key observations lead us to believe that the relative sensitivity of alphaviruses to IFN-a/b-mediated antiviral activity is a primary determinant of virulence and attenuation. Our studies with Sindbis virus (SB) have revealed that this alphavirus with little or no ability to evade or antagonize mammalian IFN-a/b is extremely attenuated in mice whereas, a mouse-virulent alphavirus, such as Venezuelan equine encephalitis virus (VEEV), is relatively much more resistant to the antiviral activity of IFN-a/b. Mutants of VEEV with increased sensitivity to IFN-a/b are attenuated in mice. As EEEV also remains virulent in the face of a functional IFN-a/b response, and alphavirus virulence appears to be strongly correlated with IFN-a/b resistance, we hypothesize that EEEV evades and/or disables components of the IFN-a/b response. We propose to gain a better understanding of the way(s) in which EEEV overcomes the antiviral activity of IFN-a/b by comparison to SB with the goal of developing an IFN-a/b sensitive, live-attenuated EEEV strain by determining which IFN-a/b-mediated pathway(s) are antagonized or evaded by EEEV. Specifically we will determine relative to SB whether EEEV is able to: i) antagonize/resist antiviral effectors; ii) avoid/suppress induction of IFN-a/b; and/or iii) suppress IFN-a/b- mediated signaling. We anticipate that these studies will provide the foundation for future identification and disablement of EEEV-encoded product(s) that antagonize IFN-a/b activity. Anticipated product(s): Our long-term goal is the rational design of attenuated alphavirus strains with sufficient degree of attenuation to be safe for human populations via the selective inactivation of innate immune evasion properties. [unreadable] [unreadable] [unreadable]